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Background  Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in  Candida albicans . A simple molecular method for rapid detection of  ERG11  gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the  C. albicans ERG11  gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of  ERG11  mutations and their type in 25 Australian clinical  C. albicans  isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing.    Results  The RCA assay correctly identified all  ERG11  mutations in eight "reference"  C. albicans  isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an  ERG11  mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate.    Conclusion  The sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of  ERG11  polymorphisms. It showed excellent concordance with  ERG11  sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant  C. albicans  and to study the epidemiology of  ERG11  mutations. The RCA method is applicable to the study of azole resistance in other fungi.

bmc microbiology

Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing